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Carrageenans as heat stabilisers of white wine

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Carrageenan addition has been previously shown to remove proteins from wine and to heat stabilise wines, however, many types of carrageenans are now available with potentially different protein‐adsorbing properties. This study investigated a range of commercially available carrageenans added at several stages of winemaking for efficacy of protein removal, heat stability and impact on wine sensory properties. In preliminary screening trials, 11 types of carrageenan were added to a Chardonnay wine and the heat stability of the wine measured with a heat test. Three of the carrageenans successfully heat‐stabilised the wine and were included in large‐scale winemaking trials: sodium‐rich kappa (kN), potassium‐rich kappa (kK) and kappa/iota (90:10, ki) carrageenan. Each carrageenan was added at three stages of winemaking, to juice, during fermentation and to wine. All carrageenans produced heat‐stable wine regardless of time of addition. Addition of kN during fermentation also improved wine recovery compared to bentonite addition, the positive Control, although sodium concentration also significantly increased in the wine. Addition of kK‐carrageenan to either wine or clarified juice was the most effective treatment for producing heat‐stable wine with minimal impact on the sensory profile, wine lees, turbidity and concentration of metal ions compared to that of untreated Control wines. Kappa‐ and kappa‐/iota‐carrageenans can be effective at heat stabilising white wines without negative impact on sensory properties, although the filterability and concentration of metal ions of the wines can vary with carrageenan structure and time of addition. Kappa‐carrageenan, a renewable fining agent, is effective in heat stabilising wines and maybe become a useful alternative to bentonite.
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Carrageenans as heat stabilisers of white wine
S. RATNAYAKE
1
, V. STOCKDALE
2
, S. GRAFTON
2
, P. MUNRO
2
, A.L. ROBINSON
2
, W. PEARSON
3
,
J.M. MCRAE
3
and A. BACIC
1,4
1
ARC Centre of Excellence in Plant Cell Walls, School of Biosciences, University of Melbourne, Parkville, Vic. 3010,
Australia;
2
Treasury Wine Estates, Nuriootpa, SA 5355, Australia;
3
The Australian Wine Research Institute, Glen
Osmond, SA 5064, Australia;
4
La Trobe Institute for Agriculture and Food, La Trobe University, Bundoora, Vic. 3086,
Australia
Corresponding author: Dr Vanessa Stockdale, email vanessa.stockdale@tweglobal.com
Abstract
Background and Aims: Carrageenan addition has been previously shown to remove proteins from wine and to heat stabi-
lise wines, however, many types of carrageenans are now available with potentially different protein-adsorbing properties.
This study investigated a range of commercially available carrageenans added at several stages of winemaking for efficacy of
protein removal, heat stability and impact on wine sensory properties.
Methods and Results: In preliminary screening trials, 11 types of carrageenan were added to a Chardonnay wine and the
heat stability of the wine measured with a heat test. Three of the carrageenans successfully heat-stabilised the wine and
were included in large-scale winemaking trials: sodium-rich kappa (kN), potassium-rich kappa (kK) and kappa/iota (90:10,
ki) carrageenan. Each carrageenan was added at three stages of winemaking, to juice, during fermentation and to wine. All
carrageenans produced heat-stable wine regardless of time of addition. Addition of kN during fermentation also improved
wine recovery compared to bentonite addition, the positive Control, although sodium concentration also significantly
increased in the wine. Addition of kK-carrageenan to either wine or clarified juice was the most effective treatment for pro-
ducing heat-stable wine with minimal impact on the sensory profile, wine lees, turbidity and concentration of metal ions
compared to that of untreated Control wines.
Conclusions: Kappa- and kappa-/iota-carrageenans can be effective at heat stabilising white wines without negative impact
on sensory properties, although the filterability and concentration of metal ions of the wines can vary with carrageenan
structure and time of addition.
Significance of the Study: Kappa-carrageenan, a renewable fining agent, is effective in heat stabilising wines and maybe
become a useful alternative to bentonite.
Keywords: bentonite, heat stability, kappa-/iota-carrageenan, kappa-carrageenan, lambda-carrageenan, white wine protein haze
Introduction
Grape pathogenesis-related proteins, including thaumatin-
like proteins and chitinases, are the major soluble proteins
found in grape juice and are responsible for the formation
of hazes in white wine (Tattersall et al. 1997, Vincenzi et al.
2011). These haze-forming proteins survive the winemaking
process but during transportation and storage, when wine
can be exposed to unfavourable environmental conditions,
they can undergo aggregation resulting in the appearance of
a haze (Waters et al. 2005, Vincenzi et al. 2011, Van Sluyter
et al. 2015). These hazes are commercially unacceptable and
therefore the haze-forming proteins must be removed
before bottling. The current method of protein stabilisation
in commercial white winemaking is by adding bentonite, a
cheap, absorbent clay that is an efficient fining agent that
binds grape proteins and removes them through sedimenta-
tion (Muhlack et al. 2006, 2016). The use of bentonite,
however, has some negative aspects, such as loss of wine
volume, incompatibility with filtration equipment and sus-
tainable waste disposal challenges (Majewski et al. 2011).
Furthermore, the use of bentonite, particularly at high dose,
can remove phenolic substances, flavour and aroma com-
pounds and therefore cause an inadvertent loss of flavour in
the wine (Lambri et al. 2013, Dordoni et al. 2015, Vincenzi
et al. 2015). An alternative, cost-effective, fining agent
would therefore be desirable to the wine industry.
Protein stabilisation using polysaccharides, such as carra-
geenans, is well established in the milk and brewing indus-
tries (Duan et al. 2006). Previous reports of the addition of
carrageenans to white wine and grape juice for haze
stabilisation have shown that they can be effective when
added either during wine processing or to the finished wine
(Cabello-Pasini et al. 2005, Marangon et al. 2012, 2013). Car-
rageenans are naturally occurring polysaccharides extracted
from red seaweeds of the Rhophyta species(Necasand
Bartosikoya 2013) and are therefore a potential renewable
heat-stabilising agent. They are also a permitted additive to
wine in Australia, New Zealand, Europe and the USA (Food
Standards Australia and New Zealand 2017). These polysac-
charides are composed of a galactan backbone of alternating
1,3-linked-β-D-galactopyranosyl and 3,6-anhydro-D-galac-
tose residues with a molecular mass between 200 and
800 kDa and can contain sulfate ester (SO
3) groups. There
are three commercially available structural types of carra-
geenans, namely kappa(k-, one SO
3residue per disaccha-
ride), iota (i-, two SO
3residues per disaccharide) and
lambda (λ-, three SO
3residues per disaccharide). These sul-
fate ester polysaccharides are widely used in industrial
doi: 10.1111/ajgw.12411
© 2019 Australian Society of Viticulture and Oenology Inc.
Ratnayake et al. Carrageenans as heat stabilisers of white wine 439
applications in which kappa-carrageenans (G4S-DA repeat)
and iota-carrageenans (G4S-DA2S repeat) are employed for
their gelling properties and lambda-carrageenan (G2S-
D2S6S), a non-gelling carrageenan, as a thickening agent.
Strong interactions between the sulfate ester groups of
neighbouring helices, mediated by potassium (K
+
) and cal-
cium (Ca
2+
) ions, are responsible for stabilising the three-
dimensional structures of kappa- and iota-carrageenans,
respectively (Janaswamy and Chandrasekaran 2002). Carra-
geenans naturally occur as a K
+
salt, although they can be
modified to the sodium (Na
+
) salt through a patented ion
exchange process (Campo et al. 2009, Trudsoe 2010).
Kappa-carrageenan as the Na
+
salt is soluble in cold water
and delays the aggregation of the helix structure thereby
forming a hard gel.
Carrageenan has previously been shown to heat stabilise
white wines (Marangon et al. 2012, 2013); however, the
impact of different carrageenan structures and times of addi-
tion on winemaking processes and wine properties is
unknown. In the current study, an initial screening was
undertaken of several commercially available carrageenans
of varying structure types in a Chardonnay wine. From this
initial screening process, the carrageenans that were identi-
fied as more effective at heat stabilising wines were selected
for pilot winemaking trials. The impact of the addition to
juice, wine or fermentation of different carrageenans,
including kappa-carrageenans with either Na
+
,K
+
ions or
blended with iota-carrageenan, was explored in pilot-scale
winemaking for heat stabilisation, fermentation time, wine
recovery, clarity and filterability, concentration of metals
and sensory profiles.
Materials and methods
Heat-stabilising and clarification agents
Carrageenans with different structures were obtained from
CPKelco ApS (Lille Skensved, Denmark), Cargill Australia
(Melbourne, Vic., Australia) and Herbstreith & Fox
(Neuenbürg, Germany). Low-methyl citrus pectin was
obtained from Herbstreith & Fox and bentonite (SIHA
Active G, 0.5% sodium, 0.8% calcium) was from
E. Begerow, Langenlonshiem, Germany. Details of the car-
rageenan batches are shown in Table S1. Clarification agents
tested prior to carrageenan addition included pectinase
enzymes (polygalacturonase, VinoClear, Novozymes, Copenha-
gen, Denmark), a chitosan-based clarification agent [Qi-Up,
comprising tartaric acid (47%), pea protein (33%) and chitosan
(20%), Institut Oenologique de Champagne, Mardeuil,
France], and a combined enzyme treatment [referred to as E2,
50:50 blend of endo-β-glucanase (Viscozyme) and cellulase
(NS26215) enzymes (Novozymes, Copenhagen, Denmark)].
Stock solutions for protein removal
Stock solutions of carrageenans (10 g/L) were prepared for
addition to either juice, ferment or wine to remove proteins.
Stock solutions were prepared in the same media as the
sample to be treated (juice, ferment or wine) and the vol-
ume required was removed from the vessel, mixed with the
required dose of carrageenan and returned to the original
vessel to avoid dilution effects. Stock solutions were freshly
prepared by slowly adding carrageenan (2 g) to either juice,
ferment or wine (200 mL) while mixing using a mixing stir-
rer (ES Overhead Stirrer with stirring shaft with propeller,
Velp Scientifica, Usmate, Italy) for at least 2 min until all
material was in solution. Bentonite stock solutions (250 g/L)
were prepared with water and included in the preliminary
heat stabilisation trials and large-scale winemaking trials as
positive Controls.
Carrageenan characterisation
Carrageenans were characterised for water- and wine-
solubility using viscosity index measurements. Carrageenan
solutions (1.5% w/v; 15 g/L) were prepared in both water
and wine (Chardonnay) and the viscosity index of each
solution was visually rated on a scale of 16, where 1 = low
viscosity (liquid), 6 = high viscosity (solid). To determine
total carbohydrate concentration, each carrageenan
(200 mg) was dissolved in H
2
O to which four volumes of
absolute EtOH were added. After 18 h at 4C, the solution
was centrifuged, the pellet washed twice with EtOH, ace-
tone and methanol, dried under vacuum and weighed. Car-
rageenan structures were elucidated using linkage
composition analysis (Stevenson and Fumeaux 1991) and
sulfate ester content (Craigie et al. 1984), as well as
1
H
NMR spectroscopy (D
2
O, 80C, Bruker Avance III 600 MHz
spectrometer with cryo-probe, Bruker Daltonics, Bremen,
Germany) as a single-step analytical method to identify dif-
ferent types of carrageenan (Figure S1 and Table S2). Carra-
geenans have been characterised extensively with
1
H-NMR
and the diagnostic chemical shifts for the key structural
units are published (Van de Velde et al. 2002). Molecular
size distribution (data not shown) was determined using
size-exclusion chromatography with an Agilent 1100 HPLC
(Agilent Technologies, Santa Clara, CA, USA) with a
Shimadzu RID-10A refractive index detector (Shimadzu,
Kyoto, Japan), GRAM 1000 Å column (PSS Polymer, Mainz,
Germany) at 80C, and dimethylsulfoxide with 0.5% lith-
ium bromide isocratic solvent system, 0.3 mL/min flowrate.
Structures were found to be similar to the product specifica-
tions, as expected, and batches of the same type of carra-
geenan were compositionally the same regardless of sample
code or production year.
Heat-stabilising trials
The efficacy of different carrageenans in producing heat-
stabilised wines was assessed in preliminary heat-stabilising
trials with a Riverland Chardonnay wine [Treasury Wine
Estates (TWE), Nuriootpa, SA, Australia]. Carrageenan stock
solution (10 g/L in wine) or bentonite stock solution
(250 g/L in water) were added to wine samples (150 mL
each in triplicate) to give a final concentration between 0.2
and 1.4 g/L in 0.2 g/L increments. Samples were then
shaken to mix and kept overnight at room temperature
before filtering at 0.45 μm (Ultipor N66, Pall Corporation,
New York, NY, USA) and assessing the heat stability of each
sample as previously described (McRae et al. 2018). The
concentration of each heat-stabilising agent that produced a
change in turbidity before and after heat treatment (80C
for 2 h, 20C for 3 h) of <2.0 nephelometric turbidity units
(NTU) (Hach model 2100P nephlometer, Loveland, CO,
USA) was considered the recommended dose. Carrageenans
that heat-stabilised the Chardonnay wine at a concentration
less than 1.4 g/L were investigated further in large-scale
trials.
The concentration of heat-stabilising agent required to
produce heat-stable wines for the large-scale trials was also
assessed using bench heat-stabilising trials. The dose for
wine addition was determined as described above with addi-
tion of either carrageenan or bentonite at 0.2 g/L across the
range of 0.21.4 g/L. The dose that gave <2.0 ΔNTU before
© 2019 Australian Society of Viticulture and Oenology Inc.
440 Carrageenans as heat stabilisers of white wine Australian Journal of Grape and Wine Research 25, 439450, 2019
and after heating (80C for 2 h, 20C for 3 h) was selected.
For juice addition, carrageenan was added in the same man-
ner as with wine prior to heating, and the dose was deter-
mined as the carrageenan concentration that produced the
lowest NTU after heating, to account for the interference of
sugars and juice solids in the test. The addition rate to the
fermentation was assumed to be the same as that for juice.
Preliminary clarification trials
Unclarified Sauvignon Blanc grape juice (2015 and 2016,
Adelaide Hills, SA, Australia) was provided by TWE. For the
clarification trials, juice was divided into batches for the
three clarification treatments and two Controls (20 L each
in triplicate) (Figure S2). For Treatment 1 (T1, chitosan
treatment), juice was first settled (0C, 24 h), carrageenan
stock solution added (10 g/L kappa-:iota- in 2 L grape juice
to give 1 g/L in 20 L), further settled for 24 h and a
chitosan-based clarification agent added (10 g/hL). Treated
juice was then allowed to settle a further 24 h prior to
racking. For Treatments 2 and 3 (T2 and T3), pectinase
(3 mL/hL) was added to juice and allowed to settle for 24 h
prior to carrageenan addition. For T2 (E2 enzyme treat-
ment), a 50:50 mixture of endo-β-glucanase and cellulase
(E2 enzymes, 60 mg/L, 15C) was added 24 h after carra-
geenan addition and allowed to settle for a further 24 h
prior to racking. For Treatment 3 (T3, pectinase treatment),
no further additions were made. The time between addition
of carrageenan and enzyme was to minimise any potential
interference of each treatment on the efficacy of the other.
Pectinase was added prior to carrageenan to prevent interac-
tions between juice solids and carrageenan. Chitosan and E2
were added after carrageenan to assess the efficacy of these
treatments in aiding the removal of protein-rich carra-
geenan prior to filtration. Controls included juice with either
carrageenan treatment only (C1) or juice with no treatment
(C2). All treated and Control juices were then fermented
(in triplicate) with Saccharomyces cerevisiae var. bayanus
(Maurivin PDM, Mauri Yeast Australia, Sydney, NSW,
Australia) at 15C to dryness (residual sugar <2.0 g/L).
Potassium metabisulfite was added (to approximately
30 mg/L free SO
2
) and wines were then settled (0C, 24 h),
cold-stabilised (2 g/L potassium tartrate, 0C, 21 days), fil-
tered using crossflow filtration (0.4 μm; Advanced Metallur-
gical Solutions, Lonsdale, SA, Australia) followed by
0.45 μm cartridge filtration (Ultipor N66 Pall Corporation)
and bottled in 750 mL green claret bottles (O-I Asia Pacific,
Melbourne, Vic., Australia). Samples were taken before and
after crossflow filtration for the filtration analysis and after
bottling for sensory and post-bottling analyses.
Large-scale heat-stabilising trials winemaking protocol
For the large-scale fining trials, Sauvignon Blanc grape
juices were sourced from the Adelaide Hills region (South
Australia) in 2015 (Wine 1) and 2016 (Wine 2), and stored
at 0C prior to use (up to 3 days). Wine 1 was produced
from unclarified juice and Wine 2 was produced from juice
treated with pectinase (0.5 g/L, Ultrazym, Novozymes) in
line with the outcome of the preliminary clarification trials
described above. Juice (2000 L) was mixed to the same tur-
bidity and transferred to 50 L fermentation vessels (48 kg
juice in each vessel). Juice was cold-settled (0C, 48 h),
racked into clean vessels (turbidity <200 NTU, Hach nephe-
lometer), and fermented to dryness (S. cerevisiae, PDM,
15C) in triplicate. Diammonium phosphate (200 mg/L) was
added to each ferment after the initial 2Be decrease.
Carrageenan stock solutions, kappa-carrageenan with K
+
ions (kK), kappa-carrageenan with Na
+
ions (kN) and kappa-
(90%)/iota-(10%) with K
+
and Na
+
ions (ki), were prepared
as described above and added to juice prior to cold-settling
(juice addition), to ferment after a decrease of 23Be (fer-
ment addition) or to wine after fermentation and potassium
metabisulfite addition (wine addition) (Figure S3). Doses for
additions were determined using fining trials as described
above for the preliminary heat-stabilisation trials. Bentonite
(1.5 g/L final concentration) was added to wine for the posi-
tive Controls, wines without any additions were the nega-
tive Controls. After each addition, either the carrageenan or
bentonite was mixed to ensure homogeneity and dry ice
was added to the vessels during mixing to minimise oxygen
exposure.
The lees volume was determined by measuring the mass
of vessels before filling and after racking. Before 0.45 μm fil-
tration, Wine 1 was pad-filtered (Z6 nominal pad filter,
IMCD Australia, Mulgrave, Vic., Australia) and then passed
through two cartridge filters (1.0 μm, absolute Pall Profile II,
Pall, Cheltenham, Vic., Australia, and then 0.45 μm, abso-
lute Ulitipor N66 Nylon) whereas Wine 2 was filtered using
a titanium cross flow (0.4 μm, nominal, Advanced Metallur-
gical Solutions) then filtered using a nylon cartridge filter
(0.45 μm, absolute Ultipor N66 Nylon, Pall). Wines were
bottled into clear punted claret bottles (750 mL, stock code
30143, O-I Glass, West Croydon, SA, Australia) and sealed
with Saran-tin screw caps (standard matte black stelvin,
Orora Closures, Dudley Park, SA, Australia). Samples were
collected before the 0.45 μm filter for filterability analysis,
before filtering and after bottling for protein analysis, and
post-bottling for wine composition and sensory analyses.
Wine filterability and membrane analysis
The filterability of wines was measured using a filterability
test as per Blue H
2
O filtration protocol (BH Technologies
2015). Wine samples were taken prior to absolute 0.45 μm
filtration and post 1 μm cartridge or after nominal 0.4 μm
crossflow-filtration. Wine (approximately 700 mL) was
added to a stainless-steel chamber and filtered through a
disc filter [0.45 μm polyethersulfone membrane, Millipore
Express] under pressure (200 kPa) and collected in a mea-
suring cylinder. For the preliminary clarification trials, the
time taken to filter 200, 400 and 600 mL of wine was
recorded to calculate the filterability index (FI
v
)
(Equation 1), where FI
v
> 20 was indicative of filtration
issues. For the large-scale trials, the filterability index (FI
T
)
was calculated based on time, whereby the collected volume
was measured at 30 s intervals up to 180 s (Equation 2),
where FI
T
> 2.0 is deemed a fail. Analyses were in triplicate.
FIV=T400 2T200 ð1Þ
FIT=V90 V30
V180 V120 ð2Þ
Wines showing filterability issues in the filterability test
were investigated further using scanning electron microscopy
(SEM) of the post-test filter membrane (Wilson and Bacic
2012). Briefly, square pieces of the filters (approximately
1 cm) were fixed in 2.5% glutaraldehyde in phosphate buff-
ered saline (PBS) for 2 h at room temperature. The filter
pieces were rinsed in PBS (3 ×20 min), dehydrated by
© 2019 Australian Society of Viticulture and Oenology Inc.
Ratnayake et al. Carrageenans as heat stabilisers of white wine 441
soaking (20 min) in solutions of increasing concentration of
ethanol in water at 10, 30, 50, 70 and 90% (v/v) ethanol
before soaking in 100% ethanol (3 ×1 h). The dehydrated
filter samples were dried in a Balzers CPD030 critical point
drier (BalTec, Pfäffikon, Switzerland) and then mounted flat
onto aluminium SEM stubs using double-sided carbon tape.
The samples were then gold coated in a Dynavac Xenosput
sputter coater (ProSciTech, Kirwan, Qld, Australia) and
imaged in a Philips XL30 Field-emission scanning electron
microscope (FEI Company, Hillsboro, OR, USA) at 2.0 kV.
Digital images were captured at a magnification of 35 000×
with a resolution of 1424 ×1024 pixels.
Wine and juice composition
Analysis of wine composition included alcohol concentra-
tion, pH, TA, volatile acidity (VA), glucose/fructose concen-
tration, free and total SO
2
and metals (Iland et al. 2012).
Sugar concentration in grape juice was measured by refrac-
tometry (Brix) with an Atago WM-7 digital handheld
refractometer(Atago, Tokyo, Japan) and by densitometry
(Baumé) with an Anton Paar DMA 35 densitometer (Anton
Paar, Graz, Austria). Protein concentration of the juice and
wine samples was determined using Bio-Rad Protein Assay
Kit (Bio-Rad Laboratories, Hercules, CA, USA). Protein was
quantified using Coomassie Brilliant Blue G-250 dye (Bio-
Rad Laboratories) with a calibration curve of bovine serum
albumin as per manufacturer instructions.
Sensory analysis
For the preliminary clarification trials, sensory analysis was
conducted using a balanced reference triangle test con-
ducted in accordance with ISO 4120-1983(E) (Standards
Association of Australia 1988) to determine any perceptible
differences between wines produced using carrageenan
treatment with or without pectinase addition. Samples were
presented to panellists in 30 mL aliquots in three-digit-
coded, covered ISO standard wine glasses at 2224C, in iso-
lated booths under colour masking sodium lighting, with
randomised presentation order across judges. Thirty-two
experienced, screened and qualified assessors evaluated the
wines for aroma and palate and selected one sample in the
set that was different from the other two samples. Data
were collected using Fizz 2.47B software (Biosystèmes,
Couternon, France) and statistical significance was deter-
mined using a binomial model (Standards Association of
Australia 1988).
For the large-scale carrageenan fining trials, descriptive
sensory analysis (Siebert et al. 2018) was used to evaluate
any differences in sensory attributes of Sauvignon Blanc
wine treated with the kN, ki and kK carrageenans at three
stages of winemaking. Appropriate descriptive terms were
developed by panel assessors during three training sessions
and used for rating in a practice session and in the formal
sessions, which were conducted over 3 days (Table 1). In
the formal sessions, panellists assessed 24 wines (eight treat-
ments in triplicate) twice using a modified Williams Latin
Square incomplete random block design generated by Fizz
2.47B software. Aliquots of each wine (30 mL), were pres-
ented in random order in ISO standard wine glasses (coded
and covered) at 2224C in isolated booths under daylight-
type lighting. A rest of 90 s was enforced between samples
and there was a minimum rest of 10 min between sets.
Wines were assessed for appearance, aroma and palate.
Each attribute was rated for intensity using an unstructured
15 cm line scale from 0 to 10, with lowand highratings
given at 10% and 90%, respectively.
Statistical analysis
Sensory data were acquired and panel performance was
assessed using Fizz Senstools (OP&P, Utrecht, The Nether-
lands) and PanelCheck (Nofima, Tromsø, Norway) as
described previously (McRae et al. 2017). All judge
responses were assessed for consistency using ANOVA of
treatment, judge and ferment replicate results (Minitab,
Sydney, NSW, Australia) and were found to be of required
standard. Tukeys honestly significant difference (HSD)
value was calculated (P< 0.05) and principal component
analysis (PCA) (XLSTAT, Addinsoft, Paris, France) was con-
ducted using the correlation matrix of the mean values for
the treatments averaged over ferment replicates, panellists
and replicates.
The effects of adding different carrageenans during three
winemaking stages compared to the untreated and benton-
ite Control wines were determined by one-way ANOVA,
and the significance of the mean assessed by Tukeys HSD
test using GraphPad Prism statistics software (v6.04 Gra-
phPad Software, La Jolla, CA, USA).
Results and discussion
Preliminary heat stability trials
Carrageenans produced commercially have different compo-
sition, particularly relating to carbohydrate concentration
and the concentration of associated metal ions, specifically
K
+
,Na
+
or Ca
2+
ions (Table 2). Of the selected carrageenans,
kappa-carrageenans were most effective at improving heat
stability by reducing haze formation after heating. Carra-
geenans incapable of heat stabilising wines contained either
lambda- carrageenan or greater than 89% iota-carrageenan.
These data indicated that only the predominantly kappa-
containing carrageenans were capable of reducing heat haze
through the potential to effectively either remove protein
from wine or stabilise the protein in wine. A mixture of
kappa- with iota-carrageenan for one batch was effective in
heat stabilising the Chardonnay wine, whereas other blends
and types of carrageenan were less effective in improving
wine heat stability (Table 2). This demonstrated that the dif-
ferences in the polysaccharide structures had a significant
impact on the capacity to adsorb proteins from wine. Kappa-
carrageenans have a single negatively charged sulfate ester
group (SO
3) per disaccharide unit which forms a strong
double helix structure as there is less repulsion between
SO
3groups compared to that in iota- and lambda-carra-
geenans, which contain two and three SO
3groups per
disaccharide unit, respectively. Lambda-carrageenans there-
fore do not form gels. Iota-carrageenans form gels in the
presence of Ca
2+
(Necas and Bartosikoya 2013, Alba and
Kontogiorgos 2018). Potassium ions neutralise the charge
between the SO
3groups in kappa-carrageenans, because
the SO
3groups are located externally to the helix structure
and this promotes either bundling or aggregation of the
helical structure forming a three-dimensional network
(Campo et al. 2009). The aggregated helical structure can be
removed from wine by either settling or centrifugation. The
mechanism for heat stabilisation of juice/wine is likely to
involve the formation of an electrostatic complex between
the negatively charged SO
3groups of neighbouring double
helices in the kappa-carrageenan and positively charged
wine proteins that entrap and precipitate the wine haze
© 2019 Australian Society of Viticulture and Oenology Inc.
442 Carrageenans as heat stabilisers of white wine Australian Journal of Grape and Wine Research 25, 439450, 2019
proteins through sedimentation (Kara et al. 2006, Campo
et al. 2009). Based on these preliminary trials, three
carragenans were selected for larger-scale trials including
natural kappa-carrageenan (kK), ion-exchanged Na
+
kappa-
carrageenan (kN) and a kappa-/iota-carrageenan blend (ki)
(Table S1).
Viscosity of carrageenans
The composition of each carrageenan determines its relative
solubility in water and wine and may relate to the efficacy for
removing proteins to heat stabilise wine. Carrageenans with a
lower viscosity index can be readily mixed using standard win-
ery equipment and may be more effectively dispersed. Higher
viscosity carrageenans can also be processed with standard
winery pumps or agitators and will facilitate protein removal
by more efficient settling, but may be more difficult to initially
disperse. Natural carrageenans contained more K
+
than Na
+
and were less soluble in water than wine. This is most likely to
be due to the presence of other ions in wine that aid carra-
geenan solubility (Leske et al. 1997). The solubility of carra-
geenans depends on both their concentration of sulfate ester
groups and the associated ionisable cations (Na
+
,K
+
,Mg
2+
and
Ca
2+
), including the presence of other solutes in the media
(Campo et al. 2009). The proportion of sulfate ester groups
and equilibrium of cations and other solutes in solution deter-
mines the viscosity of the solution and the strength of the gel.
Carrageenans that had been ion-exchanged with Na
+
, referred
to as cold-stable carrageenans, had substantially lower K
+
con-
centration and greater Na
+
concentration compared to that of
natural carrageenan. This led to greater solubility in water but
reduced solubility in wine, potentially due to the influence of
the cation on the three-dimensional structure of carrageenans
in liquid (Campo et al. 2009). In solutions such as wine, ion-
isable cations and soluble solutes either disturb the formation
Table 1. Sensory descriptive analysis attributes, definitions and standards.
Attribute Definition/synonyms Standard
Appearance
Yellow colour intensity Intensity of the yellow colour
Aroma
Overall fruit intensity aroma Intensity of the fruit aromas in the sample
Passionfruit Intensity of the aroma of passionfruit 1 tsp Passionfruit pulp (John West)
Pineapple Intensity of the aroma of pineapple 4 ×2 cm Cubes fresh pineapple, canned
pineapple juice (1 tsp, Golden Circle)
Stone fruit Intensity of the aroma of stone fruits: peach, apricot
both fresh and dried, loquat
One can of Goulburn Valley apricots and
peaches
Lemon Intensity of the aroma of lemon 1 ×2 cm Piece of lemon, lemon rind
Confection Intensity of the aroma of confection: banana lolly,
musk
1×Banana lolly and 1 ×musk lolly
Floral Intensity of the aroma of flowers: violets and
blossoms
100 mg/L Linalool (40 μL), 200 mg/L 2-phenyl
ethanol (25 μL)
Green Intensity of the aroma of green grass, green leaves,
stalks, green capsicum, green beans
Freshly picked grass and half a chopped fresh
green bean
Box hedge Intensity of the aroma of box hedge Fresh box leaves
Flint Intensity of the aroma of flint, wet stones, metals,
toast
20 μL of 1 mg/L benzyl mercaptan
Sweaty/Cheesy Intensity of the aroma of sweat, cheese, blue cheese,
cheddar cheese, body odour, sour milk, raw meat
100 μL Mix of hexanoic acid and isovaleric acid
Pungent Intensity of the aroma and effect of alcohol 4 mL Ethanol
Palate
Overall fruit intensity palate Intensity of the fruit flavours in the sample
Tropical Intensity of the flavour of tropical fruits; pineapple,
passionfruit, melon
Stone fruit Intensity of the flavour of stone fruits; peach, apricot,
nectarine
Lemon Intensity of the flavour of lemon including aftertaste
Green apple Intensity of the flavour of green apple including
aftertaste
Green Intensity of the flavour of green grass, green
capsicum, stalks, herbal
Viscosity The perception of the body, weight or thickness of the
wine in the mouth. Low, watery, thin mouth feel;
high, thick mouth feel
Oily The perception of oiliness in the mouth
Sweet Intensity of sweet taste in the mouth including
aftertaste
Acid Intensity of acid taste in the mouth including
aftertaste
Hotness The intensity of alcohol hotness perceived in the
mouth, after expectoration and the associated
burning sensation; low, warm; high, hot
Astringency The drying and mouth-puckering sensation in the
mouth; low, coating teeth; medium, mouth coating
and drying; high, puckering, lasting astringency
Bitter The intensity of bitter taste perceived in the mouth, or
after expectoration
Fruit after taste The lingering tropical or stone fruit flavour perceived
in the mouth after expectorating
© 2019 Australian Society of Viticulture and Oenology Inc.
Ratnayake et al. Carrageenans as heat stabilisers of white wine 443
of the gel or, if formed, is not as stable. The higher viscosity in
wine of ion-exchanged compared with natural carrageenan is
likely to be the result of the complex matrix of wine suggesting
that the ionic composition and other soluble solutes in
wine/juice are likely play a role in affecting its structure (Kara
et al. 2006). A comprehensive study of both types of kappa-
carrageenan salts is needed to fully understand the differences
in solubility.
Effect of juice clarification agents on wine filterability after
carrageenan treatment
Carrageenan is a polysaccharide that can potentially hold
juice solids in solution and adversely affect wine filterability.
The relative filterability of wines produced after carrageenan
addition to juice was assessed by juice clarification trials
(Table 3). The addition of ki carrageenan to grape juice
reduced the filterability of the resulting wine (C2) tenfold
compared to that of the untreated Control wine (C1).
Enzyme addition to carrageenan-treated wine, including
pectinase (T3) and the blend of endo-β-glucanase and cellulase
(E2) enzymes in conjunction with pectinase (T2), significantly
improved filtration compared to that of the Controls. Pectinase
addition (T3) also improved the heat stability of the wine com-
pared to the carrageenan-treated Control wine although the
enzyme mixture (T2) reduced heat stability and increased
haze. The cause of this haze is unclear and warrants further
investigation. The use of a chitosan-based clarification agent
(T1) improved wine heat stability compared to that of the
Control wines although filterability was adversely affected.
These results indicated that clarification of juice with pectinase
addition prior to carrageenan treatment will improve wine fil-
terability. Sensory analysis using the triangle test indicated that
there was a significant (P< 0.05) difference between wines
produced using carrageenan treatment alone or carrageenan
treatment with pectinase addition (data not shown). This dif-
ference, though not necessarily negative, was taken into con-
sideration for the large-scale trials.
Influence of carrageenan structure and addition time on
winemaking and filterability
Large-scale heat stability trials were conducted to assess the
effect of carrageenans added to juice, during either
Table 2. Composition of different batches of carrageenans, including type, carbohydrate concentration and cation concentration (provided by suppliers), as
well as the relative solubility in water and in wine and the heat stability of a Chardonnay wine after treatment with each carrageenan at the stated dose.
Viscosity index
Type Batch No.CHO (%) K
+
(mg/g) Na
+
(mg/g) Ca
+
(mg/g) H
2
O Wine Dose (g/L) Heat ΔNTU
κ-A978 97 91 6 ND 6 1 1.2 2.0 0.1
κ-A371 96 91 6 0.05 6 1 1.0 2.2 0.2
κ-B1R 95 100 ND ND 6 1 1.2 2.5 0.1
κ-S1263 ND 26 16 33 6 ND 1.0 4.5 0.1
κ-(CS) A533 98 1.1 55 0.9 4 3 1.0 1.8 0.2
κ-(CS) A980 ND 1.1 55 0.9 4 3 1.2 3.1 0.3
κ-(SR) A369 88 85 11 5 2 ND 1.0 50.0
κ-/ι-(90:10) B2 96 100 ND ND 6 1 1.0 1.9 0.0
κ-/ι-(90:10) B3R 98 100 ND ND 6 1 1.2 3.4 0.4
κ-/ι-(90:10) SBe ND 43 51 ND 6 ND 1.0 4.4 0.65
κ-/ι-/λ-A532 96 23 63 1.6 4 ND 1.0 5.8 0.6
λ-A529 96 14 57 ND 6 ND 1.0 12.8
λ-S3889 ND 38 7 ND 3 ND 1.0 26.6
λ-A368 96 18 50 ND 5 ND 1.0 36.6
λ-/ι-(90:10) SBe ND ND ND ND 3 ND 1.0 43.6
ι-S4014 ND ND ND ND 6 ND 1.0 22.9
ι-(SR) A370 89 112 9 ND 2 ND 1.0 36.2
ι-/ ν-A367 98 43 51 ND 4 ND 1.0 17.8
ι-/ν-(CS) A372 97 6 80 ND 4 ND 1.0 30.3
Heat stability of the untreated Chardonnay wine was 34.6 ΔNTU; wines with <2.0 ΔNTU were considered heat stable. Results are shown as the mean of tripli-
cate analyses 1 SD except where indicated. Values without SD indicate an initial NTU >2.0 and therefore the heat test was not repeated. Batch numbers
include the following supplier codes: A, CP Kelco ApS; B, Cargill Australia; S, Sigma. Heat stability results produced by the carrageenans that were selected for
further trials. CHO, carbohydrate as total polysaccharide expressed as a % (w/w); CS, cold soluble; ι-, iota-carrageenan, λ-, lambda-carrageenan; κ-, kappa-carra-
geenan; ν-, nu-carrageenan; k-/ι-(90:10), commercial blended κ-/ι-(90:10) carrageenan consists of 94.4% κ-and 5.6% ι-carrageenans when measured by link-
age analysis; ND, not determined; SR, semi-refined.
Table 3. Effect of grape juice additives for the preliminary clarification trials on wine filterability indices before and after cross-flow filtration, and wine heat
stability.
Sample
number Additive
Carrageenan
treatment (g/L)
Post-settling
additive
FI pre-
crossflow
FI post-
crossflow Haze (ΔNTU)
C1 –– –64 42915313
C2 1.0 711 264 29 1 1.0 0.2
T1 1.0 Chitosan107 15 35 3 0.3 0.4
T2 Pectinase 1.0 E2 41 12 24 9 8.4 1.3
T3 Pectinase 1.0 36 6297 0.3 0.3
Results are given as the mean of six replicates +/1SD.Chitosan-based clarification agent. , no addition; C1, Control wine (no treatment); C2, wine treated
with kappa-/iota-carrageenan; E2, blend of endo-β-glucanase and cellulase; FI, filterability index; T1, flotation agent addition; T2, E2 enzyme addition with
pectinase; T3, pectinase addition.
© 2019 Australian Society of Viticulture and Oenology Inc.
444 Carrageenans as heat stabilisers of white wine Australian Journal of Grape and Wine Research 25, 439450, 2019
fermentation or to wine, on winemaking processes and
wine characteristics including filterability and heat stability.
Treated wines were compared to an unfined wine as a nega-
tive Control, and a bentonite-fined wine as a positive Con-
trol (Table 4). The required dose rate for addition during
winemaking depended on the time of addition (Table 4).
Generally, additions to juice or during fermentation were
lower than those added to wine which may be due to diffi-
culties in determining accurate heat test results in grape
juices as the reverse has been noted for bentonite additions
(Pocock et al. 2011). The amount of carrageenan required
for the ferment additions was considered the same as for
juice additions. Carrageenan addition increased the time of
fermentation by approximately 1 day when added to juice
and by around 2 days when added during fermentation
compared to the untreated and bentonite Control wines
(Table 4). A previous investigation reported that the pres-
ence of carrageenan could reduce the fermentation time
(Marangon et al. 2012) although that was not observed in
this case.
Wine lees were measured following the racking after cold
stabilisation to determine the efficacy of carrageenan in wine
recovery as compared with the bentonite and untreated Con-
trol wines (Table 4). The addition of kN to the ferment gener-
ated around half the lees volume generated by bentonite.
Wine recovery is one of the main reasons cited for finding an
alternative to bentonite (Majewski et al. 2011). Improved
wine recovery relative to bentonite is necessary for the adop-
tion of carrageenans, which are more expensive fining agents
compared to bentonite. These results indicated that kN carra-
geenan addition during fermentation can provide better wine
recovery than bentonite for wines produced from clarified
juice. Other carrageenan additions produced a similar volume
of lees as the bentonite and untreated Control wines. Unlike
bentonite, carrageenans tend to float rather than settle when
added to clear wine (Figure S4); however, mixing carra-
geenan to juice or during fermentation will cause the carra-
geenan to settle for efficient racking and filtration. Therefore
carrageenan is best added at stages in winemaking that
already generate lees for more effective settling and removal.
The potential for carrageenans to facilitate a dual role of a
clarification agent in aiding flotation and heat stabilisation
should also be explored. Wine turbidity, the measure of wine
clarity prior to filtration, was generally not significantly
altered by carrageenan addition to wine compared to that of
the untreated Controls. The exception was ki addition, which
Table 4. Effect of the addition during large-scale winemaking trials of carrageenans and bentonite to either juice, during fermentation or to wine on fermen-
tation time, quantity of wine lees, wine filterability and filterability index.
Wine and treatment
Addition
timeDose (g/L)
Length of
fermentation
(days)
Wine
lees (%)
Wine turbidity
(NTU)§Filter type
Filterability
index (FI
T
)
Wine 1
Control None 0.0 6.0 ND ND PFc 1 0
kN-J Juice 1.2 7.0 ND ND PF 7 4
kN-F Ferment 1.2 8.0 ND ND PF 16 13
kN-W Wine 1.4 ND ND ND PF 1 0
ki-J Juice 1.2 7.3 ND ND PF 3 1
ki-F Ferment 1.2 8.0 ND ND PF 10 13
ki-W Wine 1.0 ND ND ND PF 1 0
Bentonite Wine 1.5 6.0 ND ND PF 1 0
Wine 2
Control-p None 0.0 10.0 8.3ab 12.9b CFd 1 0
kN-Jp Juice 1.0 10.6 ND ND CF 1 0
kN-Fp Ferment 1.0 11.6 5.5b 4.3d CF 1 0
kN-Wp Wine 1.4 ND 7.0ab 11.9b CF 1 0
ki-Jp Juice 1.0 10.0 ND ND CF 1 0
ki-Fp Ferment 1.0 11.0 7.1ab 5.6cd CF 1 0
ki-Wp Wine 1.4 ND 10.0ab 21.2a CF 1 0
kK-Jp Juice 1.2 11.3 ND ND CF 1 0
kK-Fp Ferment 1.2 11.3 8.2ab 4.0d CF 1 0
kK-Wp Wine 1.4 ND 8.8ab 9.9bc CF 1 0
Bentonite-p Wine 1.5 10.0 11.2a 2.0d CF 1 0
Values with different lowercase letters in the same column are statistically different. Juice additions were made prior to cold-settling, ferment additions were
made after a decrease of 23Be and wine additions were made immediately after fermentation at the same time as SO
2
addition. Juice for Wine 2 was treated
with pectinase (0.5 g/L) prior to fermentation. Dose rates were determined using a heat test. §Measure of wine clarity post-racking of fining agent and gross
lees prior to filtration. Wines with a filterability index (FI)
T
> 2.0 have potential filterability issues. CF, crossflow filtration; F, fermentation; ι,iota-carrageenan;
j, juice; κ-, kappa-carrageenan; κK, potassium-rich κ;κN, sodium-rich κ; ND, not determined; p, pectinase treated; PF, pad filtration; W, wine.
Figure 1. Scanning electron micrographs (35 000×) of filter membranes
after filtering Wine 1 containing kappa-carrageenan (kN) added at different
winemaking stages. (a) juice addition [kN-J, filterability index (FI
T
)=74];
(b) ferment addition (kN-F, FI
T
=1613); (c) wine addition (kN-W,
FI
T
=10); and (d) bentonite Control (FI
T
=10).
© 2019 Australian Society of Viticulture and Oenology Inc.
Ratnayake et al. Carrageenans as heat stabilisers of white wine 445
increased wine turbidity but not to an extent that filterability
was negatively influenced. Carrageenan addition during fer-
mentation reduced wine turbidity to a similar extent as ben-
tonite, indicating that the carrageenan lees had completely
settled, and addition under these circumstances was as effec-
tive as bentonite in clarifying wines (Table 4). The addition of
both K
+
and Na
+
kappa-carrageenan to wine produced similar
turbidity to that of the untreated Control wine post-racking
whereas addition of ki-carrageenan to wine almost doubled
the turbidity. This suggested that adding carrageenan during
fermentation is a more effective strategy to reduce lees pro-
duction and to improve wine clarity.
The impact on wine filterability of carrageenan addition
at different stages of winemaking was also assessed prior to
0.45 μm filtration and bottling (Table 4). Carrageenan addi-
tion to juice or during fermentation reduced filterability in
Wine 1 with filterability indices (FI
T
) >2, suggesting poten-
tial filtration issues. Carrageenan addition to wine, however,
did not negatively impact filterability. The cause of the poor
wine filterability after carrageenan addition was investigated
further using SEM analysis of the 0.45 μm membranes
(Figure 1). After filtering kN wines, membranes showed
strands of polymeric material filling the regular membrane
pores of the filters for kN-J and kN-F (Figure 1a,b, respec-
tively). These strands were absent from kN-W wines and
the bentonite-treated wines (Figure 1c,d, respectively). This
suggested that the poor filterability may be due to an inter-
action between juice or ferment solids and the carrageenan
polysaccharides which prevented adequate settling. A previ-
ous study also indicated that carrageenan addition to wines
can reduce filterability (Marangon et al. 2013). To avoid fur-
ther filterability problems, juice for Wine 2 was first treated
with pectinase which was demonstrated in the preliminary
clarification trials to be effective in preventing filterability
issues. Pectinase promotes the cold settling of juice and
therefore was likely to improve wine clarity and filterability
(Sieiro et al. 2012). All wines treated with pectinase prior to
Figure 2. Proportion of protein removed by carrageenan treatment pre-
bottling ( ) (and post-bottling ( ) of (a) Wine 1; and (b) Wine 2 when added
at different stages of winemaking compared to the untreated Control wines.
Results are shown as the average of triplicate ferments 1 SD. Bent,
bentonite; kN-J, kappa-carrageenan with sodium ion, juice addition; kN-F,
kappa-carrageenan with sodium ion, ferment addition; kN-W, kappa-
carrageenan with sodium ion, wine addition; ki-J,kappa-iota carrageenan
blended juice addition; ki-F,kappa-iota carrageenan blended ferment addition;
ki-W,kappa-iota carrageenan blended wine addition; kK-J, kappa-carrageenan
with potassium ion, juice addition; kK-F, kappa-carrageenan with potassium
ion, ferment addition; kK-W, kappa-carrageenan with potassium ion, wine
addition;
Table 5. Impact of carrageenan addition during large-scale winemaking trials on protein concentration and heat stability post-bottling.
Sample Protein (mg/L)
Heat stability
post-bottling (ΔNTU)15 Rating
Heat stability
13 months (ΔNTU)
Heat stability
25 months (ΔNTU)
Wine 1
Control 37.5a 37.8 3.6 Fail 37.0 7.7 48.0 8.2
kN-J 2.7b 0.7 1.0 Pass 0.8 0.4 0.8 0.2
kN-F 2.5b 0.1 0.0 Pass 0.3 0.1 0.1 0.1
kN-W 3.9b 0.0 0.0 Pass 0.1 0.1 0.2 0.1
ki-J 2.9b 0.9 0.7 Pass 0.5 0.1 0.8 0.7
ki-F 2.5b 0.0 0.0 Pass 0.1 0.1 0.1 0.1
ki-W 3.5b 0.1 0.1 Pass 0.2 0.2 0.6 0.2
Bentonite 2.5b 0.0 0.0 Pass 0.1 0.0 0.6 0.5
Wine 2
Control-p 24.3a 78.0 2.9 Fail 89.9 5.2 ND
kN-Jp 6.9b 1.8 1.7 Fail 3.9 3.3 ND
kN-Fp 2.1cd 0.3 0.2 Pass 0.2 0.1 ND
kN-Wp 1.7d 0.7 0.1 Pass 1.7 1.9 ND
ki-Jp 4.5bcd 1.3 1.2 Fail 3.0 2.1 ND
ki-Fp 1.9cd 0.3 0.1 Pass 0.4 0.1 ND
ki-Wp 2.0cd 1.2 0.5 Pass 0.5 0.2 ND
kK-Jp 5.7bc 2.0 1.2 Fail 4.8 2.3 ND
kK-Fp 1.8cd 0.3 0.1 Pass 0.4 0.2 ND
kK-Wp 2.1cd 0.9 0.4 Pass 0.7 0.5 ND
Bentonite-p 0.9d 0.0 0.0 Pass 0.1 0.0 ND
Results are expressed as an average of each treatment replicate 1 SD. For protein concentration, means with a different letter are significantly different
(P0.05). Heat stability measured as the difference in NTU of a wine sample before heating (80C, 2 h) and after subsequent cooling (20C, 3 h). F, fermenta-
tion; ι,iota-carrageenan; κ-, kappa-carrageenan; κK, potassium-rich κ;κN, sodium-rich κ; J, juice; ND, not determined for these samples; p, pectinase; W, wine.
© 2019 Australian Society of Viticulture and Oenology Inc.
446 Carrageenans as heat stabilisers of white wine Australian Journal of Grape and Wine Research 25, 439450, 2019
carrageenan addition and subsequent cross-flow filtration
(nominal 0.4 μm) passed the filterability test (absolute pore
size 0.45 μm) (Table 4).
Influence of carrageenan structure and addition time on
wine heat stability
The addition of each selected carrageenan to either juice,
ferment or wine removed about 90% of the proteins present
in the treated wines compared to that of the untreated Con-
trol wines (Figure 2). The proportion of protein removed
from the wines was greater after filtration and bottling,
suggesting that some of the protein-absorbed carrageenan is
removed with filtration. Protein concentration in
carrageenan-treated wines after filtering and bottling
(Table 5) was marginally greater than the protein concen-
tration in wines treated with bentonite. This reduction in
protein contributed to the heat stability of most of the
treated wines (Table 5). The only treated wines that were
not heat stable were the addition of kN, kK and ki carra-
geenan to juice for Wine 2 with one or more replicates
producing >2.0 ΔNTU in the heat test which was higher
than the 2.0 ΔNTU required to pass the test. This may
relate to the difficulty in determining the heat stability of
grape juice. After bottling, wines were stored under cellar
conditions (16C) and the heat stability of the wines was
measured at 13 months for Wine 2 and 13 and 25 months
for Wine 1 (Table 5). Wines with either carrageenan or ben-
tonite treatment that were heat stable at bottling remained
heat stable with storage, while the untreated Control wines
were not stable. This indicated the long-term effectiveness
of carrageenan treatment.
Influence of carrageenan structure and addition time on
wine composition
Cations are known to play a part in the gelation of carra-
geenans and therefore the different metal ions present in
juice and wine are likely to influence carrageenan rheology
and viscosity (Leske et al. 1997, Campo et al. 2009). The
concentration of metals in wine is also important due to
limits for wine exports as well as influencing metal-complex
instabilities. The addition of carrageenans containing either
Na
+
or K
+
ions was also likely to influence the concentration
of these ions in each wine (Figure 3). Adding kN-carra-
geenan, which contained 55 mg/g sodium (Table 2),
increased the Na
+
concentration of the wine fourfold com-
pared to that of the Control wine (Figure 3a). The level of
Na
+
present in the kN wines was greater than the 60 mg/L
recommended maximum for export to Switzerland
(de Loryn et al. 2014). Such increase in Na
+
concentration
may prevent the commercial use of kN in export wines. The
addition of K
+
-dominant carrageenan, kK, however, did not
significantly increase the concentration of K
+
in the wines
(Figure 3b). Potassium is the most abundant metal in wine
and is a strong gelation agent for kappa-carrageenans (Leske
et al. 1997, Campo et al. 2009). The concentration of cal-
cium ions in Wine 2 following addition of ki and kK carra-
geenan was significantly higher than that in the kN-treated
wines (Figure 3c), although the concentration of calcium
ions in the carrageenan product was low (Table 2). A Ca
2+
concentration above 120 mg/L in wine may present a risk
for Ca
2+
tartrate instability (McKinnon et al. 1995). The con-
centration of copper, iron, manganese, zinc and magnesium
(Table 6) was not altered by the addition of carrageenans at
any winemaking stage to a level that would be detrimental
to either wine quality or metal instability. Wine chemical
analysis (Table 6) also indicated that the addition of
Figure 3. Concentration of (a) sodium ions, (b) potasium ions and
(c) calcium ions in Wine 1 and Wine 2 after addition of heat-stabilising
agents including Control ( ), bentonite ( ), kappa-carrageenan with sodium
ions (kN) ( ), kappa-iota carrageenan blend (ki)( ) and kappa-carrageenan
with potassium ions (kK) ( ). Results are shown as the average of different
treatment times 1 SD.
Table 6. Composition of Sauvignon Blanc wines.
Wine 1 Wine 2
Alcohol (%) 13.0 0.1 13.4 0.1
TA (g/L) 6.9 0.1 6.4 0.1
Residual sugar (g/L) 0.4 0.1 1.5 0.0
pH 3.3 0.0 3.1 0.0
Free SO
2
(mg/L) 34.7 12.0 39.2 2.0
Total SO
2
(mg/L) 120.4 16.4 116.3 1.3
Copper (mg/L) 0.21 0.03 0.02 0.01
Iron (mg/L) 1.92 0.10 0.17 0.06
Manganese (mg/L) 0.54 0.07 1.28 0.07
Zinc (mg/L) 0.83 0.05 1.44 0.04
Magnesium (mg/L) 77.9 6.9 ND
Results are shown as the mean of all treatments (untreated Control wines,
bentonite-treated wines, and wines after carrageenan addition to juice, fer-
mentation or wine) 1 SD, except where indicated. Iron concentration for
bentonite-treated wine was 0.86 mg/L and was excluded from the averaged
value. ND, not determined for these samples.
© 2019 Australian Society of Viticulture and Oenology Inc.
Ratnayake et al. Carrageenans as heat stabilisers of white wine 447
carrageenan and the delay in fermentation did not signifi-
cantly impact the basic wine parameters of alcohol, pH, TA
and VA for each wine.
Influence of carrageenan structure and addition time on
wine sensory profiles
For the large-scale trials, descriptive sensory analysis was
conducted for Wine 1 and Wine 2 after 4 months bottle-
ageing to determine the impact of carrageenan addition at
different stages of winemaking on the sensory characteristics
of the wine. For Wine 1, eight of the 27 sensory attributes
(Table 1) differed significantly (P< 0.05) due to carrageenan
type and addition time compared with a bentonite Control:
yellow colour intensity, passionfruit, floral, box hedge,
sweaty/cheesy, viscosity, astringency and bitterness
(Figure 4). In addition, the attributes flint and pungent were
found to be different at a low level of significance (P0.10).
Carrageenan-treated wines and untreated Control wines
rated higher in flavour and aroma intensity and yellow
intensity than wines treated with bentonite (Figure 4a). The
sensory characteristics of wines treated with kN carrageenan
did not vary widely with addition time and were similar in
sensory profiles to the untreated Control wines, whereas the
time of addition influenced sensory profiles for wines
treated with ki carrageenans (Figure 4b). For ki carrageenan,
addition to juice resulted in wines with more box hedge
character, whereas addition to wine produced more floral
character. Ferment addition of ki resulted in more pungent
and bitter wines. Bentonite addition to wine consistently
showed a reduction in the flavour and aroma intensity of
these wines. Wine 2 with kK carrageenan added at different
stages of winemaking yielded ten significant attributes: yel-
low colour intensity, stone fruit aroma, floral, vegetal, pun-
gent, stone fruit flavour, apple flavour, green flavour, bitter
and fruit aftertaste (Figure 5). Addition of kK to wine pro-
duced more stone fruit aroma and flavour and were less bit-
ter than the Control wines (Figure 5a). Bentonite-treated
wines were less yellow and had a lower intensity of fruity
aromas than the carrageenan-treated or untreated Control
wines. Principal component analysis (PCA) also demon-
strated substantial differences in wine sensory profiles with
carrageenan addition time (Figure 5b). Addition of kKto
Wine 2 during fermentation induced more bitter and green
characters in the wine compared to addition to wine or
juice. These results demonstrated that carrageenans added
Figure 5. Sensory analysis for Wine 2 presented as (a) a Spider plot and
(b) a Scores and loadings bi-plot for principal component analysis of
sensory attributes that varied significantly with kappa carrageenan
potassium salt (kK) addition time for heat-stabilised wine, compared with
that of bentonite-treated Control wine ( ) and untreated Control wine ( ).
Time of addition of kK includes juice addition (JA) ( ), fermentation addition
(FA) ( ) and wine addition (WA) ( ).
Figure 4. Sensory analysis for Wine 1 presented as (a) a Spider plot and
(b) a Scores and loadings bi-plot for principal component analysis of sensory
attributes that varied significantly with carrageenan type or addition time for
heat-stabilised wines, compared with that for bentonite-treated wines ( ) and
untreated Control wines ( ). Carrageenan types and time of addition include
kappa carrageenan sodium salt (kN) added to juice (J) ( ), fermentation (F)
() and wine (W) ( ) and kappa-iota blended carrageenan potassium salt
(kiK) added to juice (J) ( ), fermentation (F) ( ) and wine (W) ( ).
© 2019 Australian Society of Viticulture and Oenology Inc.
448 Carrageenans as heat stabilisers of white wine Australian Journal of Grape and Wine Research 25, 439450, 2019
to wine produce more fruity or floral characters than carra-
geenans added during fermentation or to juice.
Conclusions
Kappa-carrageenans have demonstrated a capability to pro-
duce heat-stable wine without increasing wine lees or wine
turbidity. Kappa-carrageenan with K
+
ions added to wine
was the most effective treatment for producing heat-stable
wine with minimal impact on sensory profiles, wine lees,
turbidity, filterability and concentration of metal ions com-
pared to untreated Control wines. Sensory analysis also
indicated that wines heat-stabilised with carrageenans had
more intense flavour and aroma profiles than those heat-
stabilised with bentonite. Juice clarification with pectinase
substantially improved the filterability of all wines treated
with carrageenan. The potential for carrageenans to facili-
tate a dual role as a clarification agent in aiding flotation
and as a heat-stabilising agent should be further explored
given the flotation properties of carrageenans. The results
from this study demonstrated that kappa-carrageenan, a
renewable fining agent, can be used to heat stabilise wines
and may become a useful alternative to bentonite.
Acknowledgements
The authors would like to thank Treasury Wine Estates for
donating the juice and CP-Kelco, Cargill and Herbstreith &
Fox for donating polysaccharides and ongoing support. We
would also like to acknowledge the contribution of the Wine
Innovation Cluster, a pilot winery for small-scale
winemaking, with particular acknowledgment to winemakers
Mr Michael Coode and Mr John Gledhill. We would also like
to thank The Australian Wine Research Institute (AWRI) sen-
sory panellists, Dr Leigh Francis and Ms Alice Barker for their
contributions for the sensory descriptive analysis data, Mr
Neil Scrimgeour for assistance in sensory coordination, Ms
Kate Cuijvers for filterability analyses and the AWRI Com-
mercial Services team for analysis of wine composition. This
project was supported by funds from Wine Australia, Grant
No: TWE 1301 to Treasury Wine Estates.
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Supporting information
Additional supporting information may be found in the
online version of this article at the publishers website:
http://onlinelibrary.wiley.com/doi/10.1111/ajgw.12411/abstract.
Figure S1. Schematic representation of the preliminary
clarification trials showing type of clarification agent and
addition stages for each treatment.
Figure S2. Schematic representation of the pilot scale
winemaking process and points for addition of fining agents
as described in the materials and methods section.
Figure S3.
1
H NMR spectra of (a) κ-/ι-(blended) carra-
geenan and (b) κ-carrageenan.
Figure S4. Wines treated with heat stabilising agents after
96 h settling at 4C. Each solution was mixed with a
homogeniser for 2 min before transferring to a glass mea-
suring cylinder. (a) 0.8 g/L bentonite; (b) 1.0 g/L kappa-car-
rageenan; (c) 1.5 g/L kappa-carrageenan.
Table S1. Properties of the selected carrageenan samples for
large-scale trials.
Table S2. Proton (
1
H) NMR chemical shifts for the main
structural units of commercial carrageenans.
© 2019 Australian Society of Viticulture and Oenology Inc.
450 Carrageenans as heat stabilisers of white wine Australian Journal of Grape and Wine Research 25, 439450, 2019
... These practices include the use of adsorbents [117], such as zirconium dioxide (ZrO 2 ) also known as zirconia [37,[119][120][121] carrageenan [6, 92,122], silica gel, hydroxyapatite and alumina [42], magnetic nanoparticles [123] zeolites [124,125] and dicarboxymethyl cellulose [126]. However, all of them are at the moment under investigation and therefore not allowed by the International Organisation of Vine and Wine (OIV) or by the European Union (EU) legislation for application in wine. ...
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